Enzyme products

ABSTRACT

AN ALKALINE PROTEOLYTIC ENZYME OBTAINED BY SUBMERGED AEOBIC CULTIVATION OF THE MICROORGNISM BACILLUS FIRMUS NRS 783.

F A QQ w l gw Q Sm Q\ l com NN @QQ 3 A www wuz E Aug. 6, 19h74 UnitedStates Patent() U.s.'c1. 7195-76;V 3 claims ABSTRACT F THE-DISCLOSURE Analkaline proteolytic enzyme obtained by submerged aerobic cultivationof' the microorgnisrn Bacillus jrmus NRS 783.

The present invention relates to a process for the preparation of anenzyme product, enzyme products prepared by the said process and the useof such products in cleaning agents and dehairing products. The termcleaning agents encompasses detergents as well as dishwashing agents.

lProteolytic enzymes produced lby cultivation of members of the genusBacillus in suitable nutrient media are widely used in detergents. It isimportant for the proper functioning of those en zyn1 es that they areactive in solutions of detergent, i e. at pH values in the range of from9 to 11 and inA thepresence of sequestering agents, surfactanits, and insome cases, `sodium perborate.

It is furthermore important forV the economy of the process that theenzyme can' be effectively used in as low a concentration as possible.

According to the present invention it has n ow been found that themicroorganism of Bacillus frmus, strain NRS 783, -when`cultivated undersuitable conditions Will form a proteolytic'enzyme composition which isparticularly well suited for use in detergents, Where it will result ina surprisingly high' cleaningvalue at a very low concentration. f

Bacillus frmus strain NRS 783 may be obtained from U.S. Department ofAgriculture, Agricultural Research- Service, Northern UtilizationlResearch and Development Division, 1815 N University St., Peoria, Ill.61604, U.S.A., as strain NRRL No. B 1107. f

In order to produce the proteolytic enzyme Bacillusfirmas NRS 783 iscultivated under .aerobic conditions in a nutrient medium containingassimilable carbon and nitrogen together with other essentialnutrients-the medium being composed in accordance with lthe principlesof the known art.

Suitable carbon sources are carbohydrates, such as saccharose, glucoseand starch, or,carbohydrate containing materials such as cereal grains,malt, rice and sorghum. The carbohydrate concentrationincorporatedinthai-medium mayvary widely, c g. up to 25% and down to 1'-5%, butusually8-10% Wi1l-be sui table,lthe percentages being calculated asdextrose. v The nitrogen source in the nutrient medium may be ofinorganic and/or organic nature. Suitableinorganic nitrogen sources arenitrates and ammonium salts. Among the organic nitrogen sources quite a'Vnumber are regularly used infermenta'tion processes irivlvinglthecultivation of bacteria. VIllustrative examples are soybean meal, cottonseed meal, peanutmeal, casein,` corn steep liquor, yeast extract, urea;and albumin. In addition the nutrient medium should also contain theusual trace substances. The `temperature-at which thecultivation isusually carried out is within the range normally employed for thecultivation of species of the genus Bacillus. The temperatureY ispreferably 30 to 40)` C. As the cultivation has to 9.7 by addition of10% of a 1 M solution riCe be carried out under aerobic conditions, itis necessary tomake use of artificial aeration, when growing thebacteria in fermentation tanks. The amount of air used is similar tothatused in conventional cultivation processes.

It isv generally preferred to perform the cultivation at alkaline pHvalues. This may be obtained by the addition of suitable buifers, suchas sodium carbonate or a. mixture of sodium. carbonate and sodiumbicarbonate after sterilization of the medium.

After fermentation, liquid enzyme preparations may be; produced byremoval of coarse material from the broth or, if desired, concentrationof the broth by evaporation at low temperature or by reverse osmosis.Finally preservatives may be added to the broth.

Solid enzyme-preparations may be prepared from the' puriiedand/orconcentrated broth by precipitation with salts, such as Na2SO4 orwith'water miscible solvents, such as ethanol or acetone; removal of thewater in the broth by suitable drying methods suchas spray-dryingmayalso be employed.

The invention is further illustrated in the following examples.

EXAMPLEl I Bacillus firmas NRS 783 was cultivated at 30 C. on a rotaryshaking table (220 r.p.m.) in 500 ml. baied Erlenmeyer asks containing100 ml. medium of the composition given below. After 6 days ofincubation the proteolytic activity of the culture Was determined usingthe Anson hemoglobin method, where pH of the substrate was adjusted tothe appropriate value with NaOH (J. Gen.v Physiol; 22, 79(1939)).'Unless otherwise stated in theA following, the proteolyticactivity is measured according i, to this method. y.

Composition of the medium in grams per litre:

' The starch in the medium is liquified with alpha-amylase' and themedium is sterilized by heating at 120 C. for 45 minutes. v

After sterilization, pH of the medium is adjusted to of sodiumsesquicarbonate.

' AfterV cultivation, the enzyme activity'of the" broth4 determined atpH V7.5 `was 50 AUL/1."(Anson'i`1iiits per,l

litre).

EXAMPLE 1I A pure enzyme preparation waspreparedas follows: A fermentedculture broth prepared 'as described in E xample I was centrifuged. Byprecipitatingfrointhisuper-x *tf1 natant with Na2SO4, a crudeenzymeproduct with arrenzyme activity of 2.34AUL/g.` was obtained. Thisfcrudeprecipitate wasredissolved, precipitated withacetone `and dialyzed. Theenzyme prepa'r'tion'wa's chromatographed on a CM -52.i'on exchangecolumnfelution ,being carried out with'a 103mmlar`tris-Inaleate buffercontaining 2 10`*3 molar calcium acetateA and with a1pH,of'6. 5.,Reference'is'made tothe attached drawing'frorny whichl it may be seenthat the elution with a linearNaCl gradient yielded the activity peak,which was collected infractions No. Vto. Y1.40..Thecombined.fractions`were dialyzed and freeze dried, whereby a salt free preparation with alow peptide .content andA anY activity-ofV 71.5 .AU/g". appeared.mr-

The Vcharacteristics of -the preparation-prepared according toExampleZ-Were-thendetermined: Y

The relationship between activity and pH was determined in the followingways:

(a) The substrate was urea denatured hemoglobin. The previouslymentioned Anson method was used as the method of analysis. The pHinterval between 7 and l2 was (b) The hemoglobin substrate was preparedaccording to the aforementioned article in the Journal of GeneralPhysiology, but excluding urea. When the phosphate buffer is added, someof the hemoglobin is precipitated. This precipitate is removed byfiltration and the resulting ltrate investigated, the pH of thesubstrate being adjusted with 5 is used as the substrate. The Ansonmethod was used as 1 N NaOH or 1 N HC1 tot he proper value. The pHactivthe method of analysis. The temperature interval investiity optimumwas found to be about 9 as can be seen from gated was 25-80 C., and thepH was 7 .5. The temperathe following table which, for comparisonreasons, also ture optimum was found to be 60 C. which can be seenincludes the corresponding values for substilisin Carls 10 from thefollowing table in which, for comparison the berg and an enzymeaccording to British Pat. No. 1,243, corresponding values for an enzymeprepared according 784. to British Pat. No. 1,243,784 are included:

pH 7 8 9 10 l1 12 Temperature 25 40 50 60 70 SOV Percent activity ofmaximum, sub- Percent of maximum, enzyme aetiiisin Carisberg 93 98 10096 84 66 cording to British Pat. No. Percent activity of maximum, en-1,243,784 10 26 40 70 100 30 Lyme according to British Pat. Percent ofmaximum, enzym No. 1,243,784 65 94 99 99 97 93 @Ording to the invention1e 43 61 100 30 13 Percent activity of maximum,- enme according tothemvenmn" 65 97 100 94 85 76 zo The relationship between stability andpH was deter- (b) The substrate was casein, and the method of analymmedm the followmg Way sis was a modification of the method described byTsuru he enzyfne lrelaratlon accofdmg to EXmPle H et al (Agn BOL Chem 30652 (1966)). The casein was'dissolved 1n deionized Water with addedfBritton and which is utilized in this and in the renewing tesis is Ham-'Robmsfm buffer (J- Chem- SOC 19321, il T456) 1D a C02* marsn casein Themethod was as follows: centration of 0.5 nig/i100 cm. This solution waskept for 10 minutes at 60 C. .at piH values between 4 and 12 substrate:and then analyzed according to the Anson method. The 10 ml. of a 1%solution of casein in Britton and PH stability optimum was found to beabout 6, as can Robinson Universal buier (I. Chem. Soc., 1931, be Seenfrom the fouowing table: p. 1456) adjusted to the appropriate pH value.2 ml. distilled water. 30 pH: Percent of maximum The mixture ispreheated at 40 C. in a Water bath for g 10 minutes; then 2 ml. ofenzyme solution is added and 6 9 after minutes of reaction time at 40 C.undegraded 10o protein is precipitated by the addi-tion of 5 ml. of 10%35 7 96 trichloroacetic acid. After 10 minutes standing at 40 C. '8 8,9to complete the precipitation, the precipitate is removed 9 6l byfiltration through a Whatman iiiter No. 42 and the 1.0 7 optical densityof the filtrate is read at 276 nm. against a 11 0 blank which isprepared in the same way except that tri- :12 o chloroacetic acid isadded to the substrate before the en- The temperature stability wasdetrmined in the f01 zyme lowing way: The optical density is useddirectly to express the proteo- The enzyme preparation according toExample u was lyte activity' dissolved in 01118 molar borate buffer in aconcentration ,F.or comparison reasons the PH actmty values for Sub 45of 0.2'5 mg./ 100 cm.3 and kept for 10 minutes at pH l0 mism Carlsbergand an enzyme Prepared according to Bt at different temperatures. lThetemperature interval inish Pat. b lo. 1,243,784 were determinedconcurrently. The vestgated was 30 70 C. and the Anson method was the PHaClVlY Values appeal' in the fOllOVYlIlg fable, ffm method of analysis.It lturned out that the enzyme is stable hlCll 1t appears that theenzyme accofdlng t0 the invenup to '50" C., it being understood thatstable means a non had a PH activity QPlmlml 0f 1L() and that Subtlllsln50 residual activity greater than 80%. At 70 C. the residual Carlsberghad a pH activity optimum 0f 10.5. activity was 0. Addition of 0.00'5molar CaCl?, stabilized the enzyme to the extent that the residualactivit of the PH "1"( "I 7 8 9 10 105 n 12 enzyme at 70 C. was morethan ,60%, y Percent acnvityoimaximum, The stability in 4the presence ofurea was determined ygiclgileg 47 59 7 94 10 93 5 55 in lthle followingway: d

enzyme accor g s e enzyme preparation accor ing to Example II wasPgggiizfsgxillg 39 51 57 61 62 76 10 giszosived i6 molar urea at pH 7.5in a concentration of enzyme weer ng o e nmg. l 00 cm. and left standinfor 30 minutes. Ex- 've'ntion 24 4! 55 74 89 100 62 periments wereperformed at 25, 40gand 50 C, The An. l 60 son method was the method ofanal s` Th d l che eiatiori h y 1S e resi ua a dermirlled in lioblgntgty and temperature was .ivlilties' at the different temperaturesappear below in the (a) The substrate was urea denatured hemoglobin. Theo owing table previously mentioned Anson method was used as the Temel-at method of analysis. The temperature interval investigated 65 p meC Resdual actlvlty percent was 25-80 C. and the pH was 7.5. Thetemperature op- 2'5 96 timum was found to be 50 C., as can be seen fromthe 40 68 following table: 50 i 5 Temperature: Percent of maximumlInhibition of lthe enzyme in the presence of various 25 27 70 chemicalcompounds was investigated in thefollowing 40 67 manner:

5() 100 Iri'o a solution of f2 mg. of the enzyme preparation ac- 41cording to Example II in a litre of a 10*3 molar tr-is- 18 maleatebuffer at pH 6.5 was added ethylene diamine' v9l 75 tetraacetic acidICEDTA) in an amount corresponding to a concentrationof 0.0'1 molar. Theproteolytic activity wasdetermined Yaccording to the Anson method, andit turnedv out that EDTA did not inhibit the enzyme at all. To asolution of 2 mg. ofthe enzyme preparation accogrding'to Examplegllinllitreof 1/ 15 molar phosphate bifer at, H15' was., addedi a tenfoldexcess on a molar basisoffpheuylietlyl sulphonyluoride (IPMSF). Theprotedlyticlaetivitywas determined according to the Anson methodandfitfftuned, out that PMSF exhibited a totally inhibiting ele'ct'onthe enzyme. This shows that the enzyme Y`acc..to the invention is aserine type proteinase, having serine in its aetive'center.

A solution of 0.32 mg. of the enzyme preparation according to Example`,IL- in y100 ml. of 210.2% solution of sodium tripolyphosphate, thepHfof which was adjusted to 10;,was-kept at 50,? C. for 3()A minutes andit turned out that the residual activity was 3%. It is highly surprisingthat the washing efficiency as shown hereinafter is ex cellent inspitenf the .i fact that Ithe enzyme as shown above is inactivated afterexposureto sodium tripolyphosphate, which is a common ingredient ofdetergents. No attempts have been made to explain this apparentcontradiction.

The esterase activities were determined in the following way: 1

The'anlalys's'were y'performed according to the pH stat method, .atplfI.8.0 .andat 30 C.

.k The vfol'l substrateswere used: N-acetyl-L- tyiosine ethylester-(ATEE) N-benzoyllL-"arginine 'ethylester` (BAER) N-tosyl-L-argininemethylester (TAME) Nebenzoyl-Lleucinethylester (BLEE) l.flfhecoucentrations of the substrates were as follows:

ATLEE: 0.02 molar in 0.092. molai KCl in water containing,8% dioxanBABE: 0.015 molar in 0.43 molar KCl in Water 'FA-ME: 0.02 molar in 0.092molar KCl in water containi`ng,8% dioxan BEBE: `0.0075 molarin 0.066molar KCl in 30% ethanol.

i Esterase units/Anson units Enzyme according Enzyme to Britishaccording Subtilisin Pat. to the Carlsberg 1,243,784 invention f'f TheMielztaelisy -constant isde'termined inthe following ways:,.v A'IEE `isused as substrate?Theideterrinationiscarriedout byy the pH-stat methodat pH 8.0 and at 30 C. The initial `velo`j; itieY Y areualculatedon thevbasis of the slope of the straight line portion of the curve, whichis'drawn by thetitrigraph. .1. I v .isi :determined at:A then-following,substrate concentrations S :Sg-10.022,*0.015,:00074. and 0.0037 -molarATEE. From the :Lineweaver-Burk plot'the Michaelis constant is found:

. the Michaelis constant is found:

Kml=0.2% hemoglobin The proportion between large and small peptidemolecules in the decomposition products from casein is determined in thefollowing way:

A 3% casein solution is digested completely at pH 8 the buffer is 0.05molar borate/ 0.2 molar NaCl) and at 50 C. for 3 hours by means of theenzymes listed below. The reaction mixture is gel filtered on SephadexG-25 superne. The fractions are collected and the amount of peptides ineach fraction is determined spectrophotometrically with ninhydrin.

The distribution between large and small peptides is stated below, thelarge peptides being defined as peptides with a molecular weight greaterthan 1000 and the small peptides being defined as peptides with amolecular weight smaller than 1,000.

Large peptides, as approximate percentage of total peptides Smallpeptides, as approximate percentage of total peptides Enzyme Enzymeaccording to the invention. Erlizzyrlggaccording to British Pat N Themolecular weight is determined in the following ways:

By ultracentrifugation the sedimentation constant is determined to about3.0 Svedberg. The specific volume is 0.72 cm3/g. and the diiusioncoeticient is about cm./sek.2 The approximate molecular weight Wascalculated to.26,000.

The molecular weight is also determined by gel iiltration on a columnwith the dimensions 2.5 cm. x 45 cm. packed with Sephadex G- superne.The column was calibrated with Blue Dextran 2000 (molecular weight2X106), Cytochrome c (molecular weight 13,000) and K2Cr04 (molecularweight 194). The buffer was 0.1 molar tris/ 1.0 molar NaCl, ph 8.0. Theow rate was 11 m1./hour. The approximate molecular Weight was determinedto 26,000.

The approximate value of the isoelectric point determined by movingboundary electrophoresis according to A. Tiselius was pI=1l.0.

The butter used in the determination vis described in G. L.' Miller andR. H. Golder, Arch. Biochem., vol. 29, pp. 420-423 (1950).

Other 4data in Yconnection with the enzyme preparation according toExample II were determined as follows:

means v,of an lactive centerutitration with N-transl Chem. "Socf88, p.5890 (1966)), and under the suppe# sition of a'molecular weightA of26,000, the purity of the cinnamoyl imidazole according to'Bender etal.(J. Amer.

product wasdetermined to 63% corresponding to a maxi-vl 4specificactivity Yof 114 All/gil() Example II, and the results of thesedeterminations are shown in the following.

The elementary analysis carried out on the enzyme preparation accordingto Example II showed the following results: C, 46.3%; H, 7.2%; N, 15.6%;S, 0.4%.

The amino acid composition was determined. This amino acid compositionappears from the following table, in which for comparison, the aminoacid composition for some enzymes known in the art are listed. It is tobe understood that the accuracy of the determination in connection withthe enzyme according to the invention is the normal 110%.

Enzyme according to- British Subtilisin B.amylo sac- Pat. The Carlsberg1 chariticus 2 1, 243, 784 invention Amino acid composition:

ysin 9 6 2 4 Histidin 5 5 4 6 Ammom 25 23 Arginin- 4 3 8 6 Aspartic acid28 20 25 23 Threonin-- 19 14 13 3 14 32 37 22 3 22 12 12 11 14 9 10 6 1236 25 24 30 42 27 23 32 (l 0 0 31 20 12 21 3 2 Z 12 11 7 16 12 11 16 y13 9 9 3 6 Phenylalanin... 4 2 3 2 Tryptophan,-.. 1 3 2 Molecularweight. 27, 300 22, 700 20, 000 26, 000

Distance from vboundary between upper and lower gel Amount of to middleof Enzyme enzyme, ng. protein band, cm.

Enzyme preparation according to Example 50 2. 72 Crystalline subtilisinCarlsberg 50 1. 70 Crystalline subtilisin NOVO 50 2. 06

All discs only exhibited one band, indicated a high purity of theenzymes.

As can be seen from the above table, the proportions between thedistances appearing in the last column of the table are as follows:

Distance, enzyme ace. to the invention 1 60 Distance, subtilisinCarlsberg Distance, enzyme acc. to the invention Distance, subtilsinNOVO Other acid disc electrophoresis experiments have been carried out.Although the values of the distances appearing in the last column of theabove table may rvary from experiment to experiment, it was found thatthe above mentioned proportions are approximately the same in all cases.

In order to demonstrate the extraordinary utility ofr the proteolyticenzyme as a detergent additive, some washing tests were v performed withartificially soiled fabrics.

A Enzyme The soiled fabrics used in the testrrwere EMPA 116 swatchescommonly used in washing tests. They are pro! duced and sold byEidgenssische A,Materialprfungsund Versuchsanstalt, Saint Gall,Switzerland. p v y The various enzyme preparations were evaluated bywashing tests in the laboratory by means of a onestep washing methodcarried out in a Launder-Ometer, which is a well-known standard modelwashing apparatus., j

In the procedures the detergent compositions used were formulated so asto simulate a heavy-dutyY detergent powder as used in Europe. f

The test conditions generally were as follows:

(1) Stained fabric:

size of matches 3.7 x 7.3 `cm. Number of swatches per f' "1 wash '5 3grams in total). (2) Washing conditions: l s l Apparatus Volume ofwashing solution 300 ml. Washing time 33 minutes. Temperature 25 C. to90 C. Detergent concentration 5 g./liter. Water hardness 10 Germanhardness.

LaunderfOmeter.

As the enzymes were used the previously mentioned proteolytic enzymeprepared according to Example II and as comparison an enzyme accordingto British Pat. No. 1,243,784. The pH after wash was 9.6.

The results of the above mentioned test are compiled in the followingtable. The figures are the `total final reflection Values indicated byR. The values of the differences between the reection values of theenzymatic detergents and the same detergent system withouttheproteolytic enzyme have been calculated and are indicated by AR. Theproteolytic activity is expressed in Anson units (All). F is theimprovement factor in comparison with one of the best, already knownalkaline, proteolytic enzymes.

WASHING TEST Enzyme according to the invention -s Enzyme according toBritish Pat. No. 1.

Furthermore, around ten similar washingtests have' been performed usingldifferent proteolytic preparations of technical grade with'activitiesbetween about lZand S- AU./ g. It turned out `that the inlprovcmentfactor F in all cases had a value betwen 1.70 and 2.10.

What is claimed is:

1. The alkaline proteolytic enzyme produced by submerged cultivation ofthe microorganism of Bacillus firmus, strain NRS 783 in a. culturemedium containing suitable carbon and N-sources, and recovered from thecultivation broth.

2. Procedure for preparation of an alkaline proteolytic enzyme whichcomprises cultivating the Organism of Bacillus frmus, strain NRS 783aerobically under submerged conditions, in a culture medium containingsuitable carbon and N-sources, thereafter recovering the enzyme from thecultivation broth.

3. Procedure for preparation of the alkaline proteolytic enzymeaccording to claim 2, wherein the cultivation is carried out between 30and 40 C., and at alkaline conditions.

References Cited UNITED STATES PATENTS 3/ 1973 Aunstrup et al. 195-62 7/1972 Aunstrup et al. 195--62 11/1971 Murao et al. 195-62 11/1971 Mitsugiet al. 195-66 R 4/1971 Murao 195-62 4/ 1972 Isono et al. 195-62 U.S. Cl.X.R.

